Novel type 1 diabetes vaccines, and methods of use

ABSTRACT

The subject invention provides compositions for alleviating type 1 diabetes (T1D). In preferred embodiments, the compositions comprise an effective amount of one or more antigen presenting cells (APCs) that have been pulsed with one or more bacterial isolates and/or compounds from the isolates. The bacteria used to pulse the APCs are, preferably, those that confer upon the APCs the ability to inhibit the generation of diabetes-promoting T cells. In specific embodiments, these bacteria may be, for example,  Eubacteria  or  Clostridia.  In a preferred embodiment, the APCs are dendritic cells (DCs).

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of U.S. provisional application Ser.No. 61/793,321, filed Mar. 15, 2013, which is incorporated herein byreference in its entirety.

BACKGROUND

Diabetes mellitus is a family of disorders characterized by chronichyperglycemia and the development of long-term vascular complications.This family of disorders includes type 1 diabetes, type 2 diabetes,gestational diabetes, and other types of diabetes.

Immune-mediated (type 1) diabetes (or insulin dependent diabetesmellitus, IDDM, T1D) is a disease of children and adults for which therecurrently is no adequate means for prevention or cure. Type 1 diabetes,represents approximately 10% of all human diabetes. The disease ischaracterized by an initial leukocyte infiltration into the pancreasthat eventually leads to inflammatory lesions within islets, a processcalled “insulitis”.

Type 1 diabetes is distinct from non-insulin dependent diabetes (NIDDM)in that only the T1D type 1 form involves specific destruction of theinsulin producing beta cells of the islets of Langerhans. Thedestruction of beta cells appears to be a result of specific autoimmuneattack, in which the patient's own immune system recognizes and destroysthe beta cells, but not the surrounding alpha cells (glucagon producing)or delta cells (somatostatin producing) that comprise the pancreaticislet. The progressive loss of pancreatic beta cells results ininsufficient insulin production and, thus, impaired glucose metabolismwith attendant complications.

The factors responsible for T1D are complex and thought to involve acombination of genetic, environmental, and immunologic influences thatcontribute to the inability to provide adequate insulin secretion toregulate glycemia.

The natural history of T1D prior to clinical presentation has beenextensively studied in search of clues to the etiology and pathogenesisof beta cell destruction. The prediabetic period may span only a fewmonths (e.g., in very young children) to years (e.g., older children andadults). The earliest evidence of beta cell autoimmunity is typicallythe appearance of various islet autoantibodies. Metabolically, the firstsigns of abnormality can be observed through intravenous glucosetolerance testing (IVGTT). Later, as the disease progresses, the oralglucose tolerance test (OGTT) typically becomes abnormal. With continuedbeta cell destruction and frank insulinopenia, T1D becomes manifest.

Type 1 diabetes occurs predominantly in genetically predisposed persons.Concordance for T1D in identical twins is 30-50% with an even higherrate of concordance for beta cell autoimmunity, as evidenced by thepresence of islet autoantibodies in these individuals (Pyke, D. A.,1979. “Diabetes: the genetic connections.” Diabetologia 17: 333-343).While these data support a major genetic component in theetiopathogenesis of T1D, environmental or non-germline genetic factorsmust also play important pathologic roles. Environmental factorsproposed to date include viral infections, diet (e.g., nitrosamines insmoked meat, infant cereal exposure), childhood vaccines, lack ofbreast-feeding, early exposure to cows' milk, and aberrant intestinalfunctioning (Vaarala et al. 2008). Hence, while the list of potentialenvironmental agents for T1D is large, the specific environmentaltrigger(s) that precipitate beta cell autoimmunity remain elusive.

Although pre-diabetogenic T cells (bearing TCR specificity forpancreatic islet cell related antigens) are essential for T1D onset,studies in rodent models (Shoda L K, et al 2005) and patients (MetcalfeK A, et al 2001; Redondo M J, et al 2001; Hyttinen V, Kaprio J, KinnunenL, Koskenvuo M & Tuomilehto J 2003) suggest that T1D may be prevented byinhibiting their acquisition of diabetogenic effector functions. Antigenpresenting cells, in particular dendritic cells, maintain immunehomeostasis by providing signals sufficient to activatepathogen-specific naïve T lymphocytes while being able to inducetolerance in naïve T cells specific to self-tissues and commensalbacteria. APC modulate immune responses by providing antigenpresentation, necessary co-stimulatory signals, and an appropriatecytokine environment.

A peaceful mutualism exists between resident gut bacteria and themammals in which they reside: the host provides food for the commensalbacteria, which in turn provide nutrients to the host by metabolizingotherwise indigestible food. In addition, a dynamic equilibrium alsoexists between resident gut flora and the development of the mammalianimmune system. In particular, Th17 effector functions are induced byresident commensal bacteria, and subsequently regulate the compositionof bacteria residing within the gut (rev in (Curtis M M & Way S S 2009;and Ivanov I I, et al 2008)).

Studies have shown that modulation of gut composition can alter onset ofT1D (rev. in (Vaarala O, Atkinson M A & Neu J 2008)). Moreover, it hasbeen recently demonstrated that distinct, naturally occurring microbialcommunities reside within the gut of Bio-breeding diabetes prone andresistant rats (Roesch L F, et al 2009), and within the subset of femaleNOD mice naturally resistant to T1D compared to susceptible syngeneicmice (Kriegel M A, et al 2011).

In terms of gut microbial regulation, APC prime T lymphocyte effectorfunctions, maintain mutualistic communities, while eliminating thoseperceived as pathogens. While the IL17A (referred hereafter as IL17)effector function by T lymphocytes is important in microbial gutcommunity regulation (Happel K I, et al 2005; Higgins S C, Jarnicki A G,Lavelle E C & Mills K H 2006; Murphy C A, et al 2003), the role of APCprimed IL17 production in the context of T1D, is less clear as it hasbeen correlated with both onset and resistance (Nikoopour E, et al 2010;Bending D, et al 2009; Martin-Orozco N, Chung Y, Chang S H, Wang Y H &Dong C 2009).

Notably, increased natural segregation of gut residing SegmentedFilamentous Bacteria (SFB) (Kriegel M A, et al 2011) and oral feeding ofLactobacillus johnsonii N6.2 (LjN6.2) (Valladares R, et al 2010) weresufficient to confer T1D resistance to T1D susceptible rodent strains.The resistance to T1D mediated by LjN6.2 and SFB was correlated to aTh17 bias (Kriegel M A, et al 2011; Lau K, et al 2010). Althoughdendritic cells prime naïve T lymphocytes and interact with resident gutflora communities directly (Grainger J R, Hall J A, Bouladoux N,Oldenhove G & Belkaid Y 2010), how distinct microbes can contribute toAPC priming of diabetogenic T lymphocytes effector functions is poorlyunderstood.

The NOD is a well-established mouse model of T1D, with destructiveleukocytic infiltration of pancreatic islets, followed by insulininsufficiency in >80% of female mice. The NOR mouse, a recombinantcongenic mouse strain, possesses 88% genetic identity with the NOD mouseand also develops leukocytic infiltrations within the pancreaticvasculature. However, unlike NOD mice, the leukocytic infiltrations inNOR do not typically progress to insulitis (i.e, intra-islet invasion),rendering NOR mice T1D free.

As noted above, one of the numerous factors that have been considered inthe context of unraveling the complex etiology of T1D is intestinalfunctioning, including the interaction of intestinal microflora. Thepresence of a commensal intestinal microbiota in infancy is critical andwell documented for numerous physiologic processes including growth,angiogenesis, optimization of nutrition, and stimulation of various armsof the innate and adaptive immune systems. However, similar studies inT1D are limited. In rodent models of T1D, the disease is likely todevelop under germ free conditions. Diabetes prone rats (BB-DP)subjected to cesarean derivation develop accelerated disease (Like etal. 1991). In terms of using such information to proactively modulatediabetes formation, antibiotic treatments to BB-DP rats after weaning(Brugman et al. 2006) prevents diabetes, whereas with the NOD mouse, adecreased frequency of T1D was observed with the administration ofdoxycycline (Schwartz et al. 2007). Probiotic treatment of non-obesediabetic mice (NOD) prevents the onset of T1D (Calcinaro et al. 2005;Yadav et al. 2007). Similarly, a low fat diet with Lactobacillus strainsreduced insulin-dependent diabetes in rats (Matsuzuki et al. 2007).Antibiotics can prevent T1D in diabetes-prone rats (BB-DP) (Brugman etal. 2006) and in NOD mice (Schwartz et al. 2006). The incidence ofdiabetes in NOD mice increases in a germ-free environment (Suzuki et al.1987; Wicker et al. 1987). Freund's adjuvant, which containsmycobacteria, also protects NOD mice and the BB-DP rat against diabetes(Sadelain et al. 1990a,b; McInerney et al. 1991). The specificmechanisms of how such therapies modulate disease are unclear.

Type 1 diabetes is currently managed by the administration of exogenoushuman recombinant insulin. Although insulin administration is effectivein achieving some level of euglycemia in most patients, it does notprevent the long-term complications of the disease including ketosis anddamage to small blood vessels, which may affect eyesight, kidneyfunction, and blood pressure and can cause circulatory systemcomplications.

Although knowledge of the immune system has become much more extensivein recent years, the precise etiology of T1D remains a mystery.Furthermore, despite the enormously deleterious health and economicconsequences, and the extensive research effort, there currently is noeffective means for controlling the formation of this disease.

BRIEF SUMMARY

The subject invention provides compositions for alleviating type 1diabetes (T1D). In preferred embodiments, the compositions comprise aneffective amount of one or more antigen presenting cells (APCs) thathave been pulsed with one or more bacterial isolates and/or compoundsfrom the isolates. The bacteria used to pulse the APCs are, preferably,those that confer upon the APCs the ability to inhibit the generation ofdiabetes-promoting T cells. In specific embodiments, these bacteria maybe, for example, Eubacteria or Clostridia. In a preferred embodiment,the APCs are dendritic cells (DCs).

The subject invention also provides methods for preventing or slowingthe development of T1D. These methods comprise the administration of acomposition of the subject invention, wherein the composition preferablycomprises an effective amount of one or more pulsed APCs.

In accordance with the subject invention, it has been found that APCsthat have been pulsed with bacteria strains can be used to alleviate(delay the onset of and/or reduce the severity or progression of), T1D.In specific embodiments of the subject invention, the administration ofDCs that have been pulsed with Lactobacillus strains such as L.johnsonii can prevent or delay the onset of, or reduce the progressionof, T1D in an animal model. Specially exemplified herein is the use ofLactobacillus johnsonii N6.2. Vaccination with DCs that have been pulsedwith Lactobacillus johnsonii N6.2 conferred T1D resistance to DProdents. Diabetes resistance in the DP rodents was correlated to a TH17bias within the mesenteric lymph nodes, which was associated with highlevels of IL6 and IL23.

The subject invention further provides methods to screen humangut-derived bacterial strains (in vitro) for their ability to be used inthe APC vaccine described herein. In one embodiment, human gut derivedbacterial strains, that have been found to be negatively correlated withdiabetes onset can be incubated with human dendritic cells (obtainedfrom blood or a human dendritic cell line) followed by assessing theability of the gut flora modulated dendritic cell to inhibit thegeneration of diabetes promoting T cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B. Resistance to T1D in NOR mice, compared to NOD, iscorrelated to enhanced TH17 differentiation and antigen presentingcells. A. Whole LN cells, isolated from NOD and NOR mice, were pooledand stimulated with anti-CD3. Forty-eight hours later, one half of theculture supernatant was collected from each sample and replenished withfresh media. At 72 hours each sample was pulsed with 3H thymidine,followed by assessment of proliferation by cellular incorporation of 3Hthymine 16 hours later. Collected supernatants were assessed for IFNγ,IL17A, and IL6 by ELISA as indicated. Each point within graphs shownindicates an individual mouse analyzed, with experiments performed induplicate. B. Ex vivo LN cells, isolated from NOD and NOR mice, werepooled and stained with antibodies specific for T cell and APC markersfollowed by analysis by flow cytometry. Top: Graphs showing absolutenumbers and relative frequency of CD4+ and CD8+ lymphocytes withinpooled LN from NOD and NOR mice. Bottom: Graphs showing absolute numberand relative frequency of total CD11c+, CD11b+, and B220+ APC presentwithin pooled LN cells from NOD and NOR mice. Absolute cell numbercounts for respective cell populations were obtained by multiplyinggating frequencies obtained from flow cytometry with total cell numbers.Averages were based upon a minimum of 3 mice from each lineage **p<0.01,***=p<0.005

FIG. 2A-2B. IL17 deficiency is correlated to decreased APC, but notCD4+CD2S+ Treg numbers. Pooled leukocyte cells isolated from theaxillary, brachial, mesenteric, inguinal, and cervical LN of NOD and NORmice were labeled with T cell and APC specific antibodies ex vivofoHowed by flow cytometry analysis. A) Graphs showing the absolutenumber (top) and relative frequencies (bottom) of CDS+, CD4+, andCD4+CD25+ T lymphocytes present in the LN of NOD and NOR mice. B):Graphs showing the absolute number (top) and relative frequencies(bottom) of 8220+, COI1b+, and CD11c+ leukocytes present in the LN ofNOD and NOR mice. Absolute cell number counts for CD II b+, CD II c+,and B220+ populations were obtained by gating first for theCD3-population. **=p<0.01, ***=p<0.005. Data is averaged for 3 mice perset.

FIG. 3A-3B. Lymphocyte infiltrates present within the NOR pancreasdisplay enhanced levels of TH17-related factors compared to NOD. Serialcryostat sections were obtained from the pancreases of 12 week oldpre-diabetic NOD and NOR mice in order to analyze the location andphenotype of T lymphocytes infiltrates. A. H&E stains of pancreasisolated from 12 week old NOD and NOR mice. Photos are at 20×magnification and representative of 3 mice from each category. Arrowsindicate representative islets present in the pancreas of each mouselineage B. Graphs showing the relative expression of CD3, IFNγ, IL17A,RORγt, and IL6 RNA message in 40 micron cryostat sections isolatedsequentially from H&E pancreas sections. Anti-CD3 results are shownrelative to actin, while message levels are shown relative to anti-CD3.*=p<0.05. A total of 6 mice per category were examined in duplicateexperiments.

FIG. 4A-4B. NOD mice possess reduced Th17 bias and distinct bacterialflora composition in the mesentery compared to NOR mice. A. Graphsshowing the expression of CD3, IL17A, and IL23R relative to β-actin inmesenteric lymph nodes isolated from 12 week old pre-diabetic NOD andNOR mice. Each point within graphs shown indicates an individual mouseanalyzed, with experiments performed in duplicate. B. Top, PCA analysisof weighted distances between NOD and NOR mice evaluating microbialdiversity. Each dot represents a sample and is identified based on type.Bottom, Stacked bar graph showing the most abundant bacterial speciespresent within stool samples isolated from NOD and NOR mice. The stackedbar graph depicts the genus of groups of bacteria sharing 97.5% sequencesimilarity with different abundances in NOD and NOR mice (p<0.05).

FIG. 5. Enhanced Th17 bias In NOR mesenteric LN is correlated toenhanced IL23production. Graphs showing the expression of RoRyt, IL23,and IL6 relative to f3-actin in mesenteric lymph nodes isolated from 12week old pre-diabetic NOD and NOR mice. Each point within graphs shownindicates an individual mouse analyzed, with experiments performed induplicate.

FIG. 6. Enhanced Th17 bias not observed in pancreatic LN. Graphs showingthe expression of IL6, IL23, IL17A, and IL23R, and RORyt relative to CD3in pancreatic lymph nodes isolated from 12 week old pre-diabetic NOD andNOR mice. Each point within graphs shown indicates an individual mouseanalyzed, with experiments performed in duplicate.

FIG. 7A-7B. Analysis of diversity and microbial communities in NOD andNOR mice at the phyla and genus levels. A) Bar graph showing nodifference (p=0.32) in community diversity between NOD and NOR micebased on Shannon-Weaver diversity analysis. B) Stacked bar graphsshowing the most abundant bacterial communities present with stoolisolated from 8 week old pre-diabetic NOD and NOR mice at the genus andphyla level.

FIG. 8A-8C. LjN6.2, but not LrTD1, increases APC activation in NOD mice.Splenocytes, derived from NOD or NOR mice, were cultured with gradeddoses of LjN6.2 or LrTD1 in the presence of anti-CD3. Following 48 hoursincubation, samples were analyzed through flow cytometry for APCactivation. A): Histograms showing changes in CD11b and CD11cfrequencies upon treatment of NOD in vitro leukocyte cultures withLjN6.2 (red), LrTD1(black) or CD3 (shaded). B) Graphs showing percentageof CD11b+ and CD11c+ leukocytes in NOD and NOR cells cultures incubatedwith graded doses of bacteria as indicated C) Graphs showing changes inMHC expression levels on CD11c+ and CD11b+ APC upon treatment withgraded doses of LjN6.2 (shaded square) or LrTD1 (open square).

FIG. 9A-9C. Enhanced dendritic cell maturation mediated by LjN6.2surface antigens. Splenocytes, derived from NOD or NOR mice, werecultured with graded doses of LjN6.2 or LrTD1in the presence ofanti-CD3, as in FIG. 4. Subsequent to incubation, samples were analyzedfor expression of the endocytic marker, DEC 205. A) Overlay histogramsshowing changes in DEC205 frequency on CD11c+ dendritic cells upontreatment of NOD cultures with varying doses of LjN6.2 and LrTD1. B)Graphical representations of DEC205 expression on CD11c+ dendritic cellspresent within NOD and NOR cultures upon incubation with varying dosesof LjN6.2 and LrTD1. C) BMDC, derived from NOD mice were matured in thepresence of GM-CSF, followed by incubation with LjN6.2 or beadbeaterlysed LjN6.2 bacterial cellular components. left, graph showing IL6production by BMDC incubated with either intact LjN6.2, or fractionalequivalents of nonviable lysed LjN6.2 components defined byultracentrifugation as membrane (pellet components) or cytosolic(soluble) components. Right: photo showing anaerobic growth of LjN6.2(4×105) or fractional membrane equivalent on MRS media after 48 hours.Data shown is average of at least two independent experiments depictingat least 5 individual NOD and NOR mice.

FIG. 10. Nonviable LjN6.2sU1face components mediate enhanced dendriticcell maturation. Splenocytes (4×10̂5) were isolated from C57BL/6 mice andplated in the presence of antiCD3 (4 μg/ml) and BMDC pulsed with eitherLjN6.2 or bacterial cellular equivalents of LjN6.2 membrane or cytosolicequivalents. Supernatants were removed at 48 hours for ELISA analysis.Graphs showing IL6 and I11? production in response to varyingconcentrations of LjN6.2 or cellular equivalents of membrane componentsas indicated.

FIG. 11. List of OTUs at 97.5% sequence similarity with differentrelative abundances in NOD and NOR mice and OTU correlations withcytokine message. OTUs with at least 0.1% abundance in either NOD or NORmice and are more abundant in either mouse type (p<D.OS, indicated inparentheses) or correlated with Th 17 associated factors are listed.Positive (+) and negative (−) correlations using Spearman correlationare indicated (significant correlations alpha<0.05).

DETAILED DESCRIPTION

The subject invention provides compositions for preventing and/ordelaying the onset of Type 1 Diabetes (T1D) (or reducing the severity ofT1D) wherein the compositions comprise an effective amount of one ormore antigen presenting cells (APCs). The composition can also includepharmaceutically acceptable carriers, additives, or excipients.

In preferred embodiments, the compositions comprise an effective amountof one or more dendritic cells (DCs) that have been primed with one ormore bacterial isolates such that the primed DCs, when administered to adiabetic subject (or a subject at risk for developing diabetes), inhibitthe progression of diabetes. The bacteria used to pulse the dendriticcells are, preferably, those that confer upon the DCs the ability toinhibit the generation of diabetes promoting T cells. In specificembodiments, these bacteria may be, for example, Eubacteria orClostridia. In other embodiments, the bacteria may be Lactobacillus.

The subject invention also provides methods for preventing or slowingthe development of T1D. These methods comprise the administration, to asubject with T1D or at risk for developing T1D, a composition of thesubject invention, wherein the composition preferably comprises aneffective amount of one or more of the primed DCs.

In accordance with the subject invention, it has been found thatdendritic cells that have been pulsed with bacteria strains can be usedto alleviate (delay the onset of, and/or reduce the severity orprogression of), T1D. In specific embodiments of the subject invention,the administration of DCs that have been pulsed with Lactobacillusstrains such as L. johnsonii can prevent or delay the onset of, orreduce the progression of, T1D in an animal model.

For example, in accordance with the subject invention, it has been foundthat vaccination with DCs that have been pulsed with Lactobacillusjohnsonii N6.2 conferred T1D resistance to DP rodents. Diabetesresistance in the DP rodents was correlated to a TH17 bias within themesenteric lymph nodes, which was associated with high levels of IL6 andIL23.

Lymphocytes, isolated from NOD mice, possess a reduced Th17 bias whencompared to counterparts from congenic, diabetes resistant NOR mice.This is notable since NOR and NOD mice contain significant numbers ofpotentially diabetogenic T lymphocytes, but only NOD mice proceed toT1D. The fact that congenic, diabetes resistant NOR mice possess a Th17bias in comparison to NOD mice is comparable to a recent study showingthat a Th17 bias is present within the subset of NOD mice naturallyresistant to T1D (Kriegel M A, et al 2011). Together these data suggestthat in addition to the conversion of pre-diabetogenic T lymphocytes toeither a Th2 lymphocyte or a Foxp3+ regulatory T cell phenotype,differentiation into the Th17 lineage may also inhibit the acquisitionof diabetogenic effector functions.

In accordance with the subjection invention, diabetes inhibitingLjN6.2enhanced APC maturation as denoted by induction of Th17lymphocytes, up-regulation of MHCI, up-regulation of MHCII, increasedIL6 production, and decreased surface expression of DEC205.Additionally, we found that nonviable LjN6.2 were sufficient to mediatedendritic cell maturation, likely through an interaction betweenbacterial membrane components and DEC205.

The amount of the therapeutic or pharmaceutical composition of theinvention that is effective in the prevention and/or treatment of T1Dcan be determined by a person skilled in the art having the benefit ofthe current disclosure through standard clinical techniques. The precisedose to be employed in the formulation will also depend on the route ofadministration, and should be decided according to the judgment of thepractitioner and each patient's circumstances. In one embodiment,effective doses can be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

The subject invention further provides methods to screen humangut-derived bacterial strains (in vitro) for their ability to be used inthe dendritic cell vaccine described herein. In one embodiment, humangut derived bacteria strains, that have been found to be negativelycorrelated with diabetes onset (using, for example, comparisontechniques similar to Brown et al. 2011, which is incorporated byreference herein in its entirety), can be incubated with human dendriticcells (obtained from blood or a human dendritic cell line) followed byassessing the ability of the gut flora modulated dendritic cell toinhibit the generation of diabetes promoting T cells. Of particularinterest are the microbes identified in U.S. Published Application No.US-2012-0183513-A1, which is incorporated by references in its entirety.

The bacterial strain can be a mutant having substantially the same orimproved properties or it can be a naturally-occurring variant thereofProcedures for making mutants are well known in the microbiological art.Ultraviolet light and nitrosoguanidine are used extensively toward thisend.

In another embodiment of the subject invention, the DCs can be pulsedwith antigens or other cellular components rather than the intact cell.The antigen or other cellular component could be, for example, a cellsurface molecule and would be chosen based upon the ability to conferupon the DCs the desired ability to inhibit diabetes-promoting T cells.

The DCs of the subject invention can be formulated into a vaccinecomposition according to known methods for preparing pharmaceuticallyuseful compositions. Formulations are described in a number of sources,which are well known and readily available to those skilled in the art.For example, Remington's Pharmaceutical Science (Martin E W [1995]Easton Pa., Mack Publishing Company, 19^(th) ed.) describes formulationsthat can be used in connection with the subject invention. Theformulations of the subject invention can include other agentsconventional in the art having regard to the type of formulationsdescribed herein.

The subject invention further provides a method of preventing or slowingthe development of T1D comprising administration of a compositioncomprising an effective amount of one or more DCs together with dietmodification, and/or the administration of other therapies, including,for example, immunosuppressants.

Other autoimmune conditions to which the treatments of the subjectinvention may be applied include, but are not limited to, rheumatoidarthritis, multiple sclerosis, thyroiditis, Crohn's disease,inflammatory bowel disease, Addison's disease, pancreas transplantation,kidney transplantation, islet transplantation, heart transplantation,lung transplantation, and liver transplantation.

Timing of Treatment

The therapies of the subject invention can be used to alleviate type 1diabetes.

In one embodiment, treatment is administered prior to the onset ofclinical manifestation of overt type 1 diabetes. The time ofadministration is preferably before extensive irreversible beta celldestruction as evidenced by, for example, the clinical onset of type 1diabetes.

As set forth in more detail below with respect to type 1 diabetes, thoseskilled in the art, having the benefit of the instant disclosure canutilize diagnostic assays to assess the stage of disease progression ina patient and then administer treatment at the appropriate time as setforth herein.

With regard to the early detection of type 1 diabetes, numerousautoantibodies have been detected that are present at the onset of type1 diabetes. Also, new serologic markers associated with type 1 diabetescontinue to be described. Four islet autoantibodies appear to be themost useful markers of type 1 diabetes: islet cell antibodies (ICA),insulin autoantibodies (IAA), glutamic acid decarboxylase autoantibodies(GADA), and insulinoma-associated-2 autoantibodies (IA-2A). These arediscussed in more detail below; however, the use of these markers toidentify those at risk for developing type 1 diabetes is well known tothose skilled in the art. In a specific embodiment of the subjectinvention, treatment is administered when a patient has at least oneantibody marker or, preferably, at least two of the antibody markers.

ICA serve an important role as serologic markers of beta-cellautoimmunity. Seventy percent or more of Caucasians are ICA-positive atonset of type 1 diabetes. Following diagnosis, ICA frequency decreases,and fewer than 10% of patients still express ICA after 10 years. Thegeneral population frequency of ICA is between 0.1% and 0.3%. In apreferred embodiment of the subject invention, ATG is administered priorto a decrease in ICA.

IAA occur in 35-60% of children at onset of type 1 diabetes but are lesscommon in adults. For example, in Australians with new-onset type 1diabetes, IAA were present in 90% of children less than 5 years old, in71% of 5-10-year-olds, and in 50% of 10-15-year-olds. In Britons withtype 1 diabetes, IAA were identified in 83% of children less than 10years old and in 56% of children 10 years old and greater.

IAA have been detected in several other autoimmune diseases. IAA wereidentified in 15.9% of patients with Hashimoto's thyroiditis and 13.5%of Graves' disease subjects. In another study, IAA frequencies invarious thyroid autoimmune diseases were 44% in Graves' disease, 21% inprimary hypothyroidism, and 23% in chronic autoimmune thyroiditis,compared with 40% in primary adrenal failure, 36% in chronic hepatitis,40% in pernicious anemia, 25% in rheumatoid arthritis, and 29% insystemic lupus erythematosus.

Approximately 2-3% of the general population express GAD autoantibodies.These antibodies are detected in 60% or more of new-onset cases of type1 diabetes. The IA-2A and IA-2βA general population frequencies aresimilar to GADA at 2-3%. IA-2A and IA-2βA are observed in 60% or more ofnew-onset type 1 diabetes cases.

Early biochemical evidence of beta cell injury is a decreasedfirst-phase insulin response to the administration of intravenousglucose (IVGTT). First-phase response is defined as the insulinconcentrations at +1 and +3 min following completion of an intravenousbolus injection of glucose (e.g., 0.5 g/kg). There is also adissociation in beta cell response to secretagogues: Initially theinsulin response to intravenous amino acid administration (e.g.,arginine) is preserved even while first-phase responses are deficient(Ganda, O. P. et al., 1984. “Differential sensitivity to beta-cellsecretagogues in early, type 1 diabetes mellitus,” Diabetes 33:516-521). In ICA-positive individuals eventually developinginsulin-dependent diabetes, first-phase insulin release diminishes at arate of about 20-40 μU/mL/year (Srikanta, S. 1984. “Pre-type 1 diabetes,linear loss of beta cell response to intravenous glucose,” Diabetes 33:717-720).

When beta cell mass has substantially declined to less than 50% but morethan 10% of normal, the OGTT may display abnormalities such as impairedfasting glucose (110-125 mg/dL) or impaired glucose tolerance (2-hglucose post-75-g challenge: 140-199 mg/dL). An abnormal OGTT prior tothe clinical onset of type 1 diabetes is more likely observed in youngerchildren. Frank clinical diabetes usually follows within 1-2 years ofthe onset of oral glucose intolerance. By the time acute symptoms oftype 1 diabetes develop, beta cell mass is believed to have declined byapproximately 90% or more from baseline. In one embodiment of thesubject invention, treatment is administered once oral glucoseintolerance is observed.

Most current procedures for the prediction of type 1 diabetes involveanalyses of multiple islet autoantibodies. In every such study reported,nondiabetic individuals who express combinations of islet autoantibodiesare found to be at greater risk for type 1 diabetes than individuals whoexpress fewer varieties of islet autoantibodies. In addition, the totalnumber of types of islet autoantibodies is usually more important thanthe specific combination of islet autoantibodies. In type 1 diabetessubjects, islet autoantibodies can also reappear after pancreas or islettransplantation, predicting failure to become insulin-independent (Bosi,E. et al. 2001. Diabetes 50:2464-247).

Thus, in genetically predisposed individuals, an environmental triggeror triggers are believed to initiate beta cell autoimmunity, which canbe identified by the presence of islet autoantibodies. With progressivebeta cell damage, there is loss of first-phase insulin response tointravenous glucose administration. Subsequently the OGTT becomesabnormal, followed by symptoms of diabetes and the diagnosis of type 1diabetes. Clearly the detection of islet autoimmunity can therefore beused as a predictive marker for the subsequent development of type 1diabetes.

Both in nondiabetic relatives of type 1 diabetes subjects and in thegeneral population, the detection of islet autoantibodies identifiesindividuals who are at high risk to develop subsequent type 1 diabetes(LaGasse, J. M. et al. 2002. Diabetes Care 25:505-511). Higher titers ofICA are more predictive than lower titers, and multiple isletautoantibodies are more powerful predictors than the presence of singleautoantibodies. The combination of ICA plus low first-phase insulinsecretion is possibly the strongest confirmed predictor of subsequenttype 1 diabetes as demonstrated in the DPT-1. When using singleautoantibodies, comparative sensitivities for the prediction of type 1diabetes are as follows: ICA>GADA>IA-2A>>IAA. Combination isletautoantibody assays (e.g., the simultaneous detection of GADA and IA-2A(Sacks, D. B. et al. 2001. J. Clin. Chem. 47:803-804; Kawasaki, E. etal. 2000. Front Biosci. 5:E181-E190) will likely supersede ICA testingin future testing programs.

The majority of individuals with type 1 diabetes have isletautoantibodies at the time of onset of the disease. In cases where it isdifficult to differentiate type1 from type 2 diabetes, the presence ofone or more islet autoantibodies (e.g., ICA, IAA, GADA, or IA-2A) isdiagnostic of type 1a, immune-mediated diabetes (Rubinstein, P. et al.1981. Hum. Immunol. 3:271-275). When individuals clinically present witha subtle, non-gketotic form of diabetes that may not beinsulin-requiring yet are islet autoantibody-positive, LADA isdiagnosed.

Administration and Formulation of the Vaccine

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to generate an immune response. Precise amounts of cells oractive ingredient required to be administered depend on the judgment ofthe practitioner. However, suitable dosage ranges are of the order of afew thousand cells (to millions of cells) for cellular vaccines. Forstandard epitope or epitope delivery vaccines then the vaccine may beseveral hundred micrograms active ingredient per vaccination. Suitableregimes for initial administration and booster shots are also variable,but are typified by an initial administration followed by subsequentinoculations or other administrations.

The manner of application may vary widely; however, certain embodimentsherein will most likely be delivered intravenously, subcutaneously,peritoneally, intramuscularly and vaginally or at the site of a tumor orinfection directly. Regardless, any of the conventional methods foradministration of a vaccine are applicable. The dosage of the vaccinewill depend on the route of administration and will vary according tothe size of the host.

In many instances, it will be desirable to have multiple administrationsof the vaccine, e.g., four to six vaccinations provided weekly or everyother week. A normal vaccination regimen will often occur in two totwelve week intervals or from three to six week intervals. Periodicboosters at intervals of 1-5 years, usually three years, may bedesirable to maintain protective levels of the immune response or upon alikelihood of a remission or re-infection. The course of theimmunization may be followed by assays for, e.g., T cell activation,cytokine secretion or even antibody production, most commonly conductedin vitro. These immune response assays are well known and may be foundin a wide variety of patents and as taught herein.

The vaccine of the present invention may be provided in one or more“unit doses”. Unit dose is defined as containing apredetermined-quantity of the therapeutic composition calculated toproduce the desired responses in association with its administration,i.e., the appropriate route and treatment regimen. The quantity to beadministered, and the particular route and formulation, are within theskill of those in the clinical arts. The subject to be treated may alsobe evaluated, in particular, the state of the subject's immune systemand the protection desired. A unit dose need not be administered as asingle injection but may include continuous infusion over a set periodof time. Unit dose of the present invention may conveniently bedescribed in terns of DNA/kg (or protein/Kg) body weight, with rangesbetween about 0.05, 0.10, 0.15, 0.20, 0.25, 0.5, 1, 10, 50, 100, 1,000or more mg/DNA or protein/kg body weight being administered.

Single or multiple administrations of the compositions are administereddepending on the dosage and frequency as required and tolerated by thepatient. In any event, the composition should provide a sufficientquantity of the proteins of this invention to effectively treat thepatient. Preferably, the dosage is administered once but may be appliedperiodically until either a therapeutic result is achieved or until sideeffects warrant discontinuation of therapy. Generally, the dose issufficient to treat or ameliorate symptoms or signs of disease withoutproducing unacceptable toxicity to the patient.

Materials and Methods Animals

Pre-diabetic NOD/ShiltJ and age-matched NORlltJ mice (The Jacksonlaboratory, Bay Harbor, Me.) were maintained in specific pathogen-freeconditions at the Association for Assessment and Accreditation forlaboratory Animal Care (AAALAC) accredited University of Florida, underthe supervision of Institutional Animal Care and Use Committee (IACUC).Pre-diabetes status of NOD/ShiltJ mice was confirmed using a bloodglucose monitoring unit (Lifescan One Touch) as having blood glucoselevels below 250 mgldl. Peripheral LN (axillary, inguinal, andbrachial), mesenteric LN, pancreatic LN, pancreas, and spleens wereextracted from each mouse for in vitro or ex vivo analysis. PrediabeticNOD/ShiLtJ and NOR/LtJ were euthanized followed by removal of peripheralLN (axillary, inguinal, and brachial), mesenteric LN, pancreatic LN,pancreas, and spleens for in vitro or ex vivo analysis.

Bone Marrow Derived Dendritic Cell Preparation

Bone marrow was removed from the femur and tibia bones of NOD mice andwashed. Progenitor cells were subsequently incubated in RPMI 1640supplemented with 10% fetal bovine serum, 1% anti-biotic/anti-mycotic,granulocyte/macrophage colony stimulating factor (GMCSF) (20 nglml) in24-well plates (I×106 cells/well). Old medium was removed and replacedwith I ml fresh complete RPMI medium containing 20 ng/ml GM-CSF every 2days. On day 8, aggregates were dislodged and transferred with completeRPMI medium into IOO-mm petri dishes at a maximum of I×107 cells/dish.At 24 and 48 hour time points following transfer, nonadherent,non-proliferating, maturing dendritic cells (BMDC) were collected fromthe dish and stored in a sterile flask.

Proliferation Assays

Lymphocyte proliferation assays were performed as previously described(1) with modifications. 4×105 whole lymphocytes were incubated with 4˜g1mL anti-CD3 (clone 17A2; eBioscience, San Diego, Calif. insupplemented RPMI 1640 (IO-040-CV; Cellgro, Manassas, Va.) containing10% fetal bovine serum (10082-147; Gibeo, Carlsbad, Calif., 1%anti-biotic/anti-mycotic (30-004CI; Cellgro) in 96-well round bottomplates. After 72 hours of incubation, cultures were pulsed with 0.5 mCieH┘ thymidine. Thymidine incorporation was measured using a BeckmanLS3801 Liquid Scintillation System.

In Vitro Cytokine Secretion Analysis

Whole lymphocytes or BMOC were incubated with 4 ˜g1mL anti-CD3, Ljohnsonii N6.2 (LjN6.2) and/or L. reuteri TOI (LrTOI) at variousconcentrations as indicated. At 48 hours, 100 ˜IL of supernatant wasremoved from each well and replenished with fresh medium as previouslydescribed (2). Cytokine EllSAs were subsequently performed on harvestedsupernatants. ELISA kits were purchased from BO Bioscience: anti-IFNi′(555138; BO Biosciences, San Diego, Calif., and anti-IL6 (555240).Capture mAb (555068) and detection mAb (555067) for IL 17A werepurchased from BO Biosciences. Cytokine standard for IL-17A waspurchased from eBioscience (14-8171-80).

Flow Cytometry

Single cell suspensions of pooled Lymph nodes (LN) (axillary, inguinal,brachial, mesenteric, and superficial cervical), and spleen were stainedwith the following mAbs for flow-cytometric analysis: anti-004-PacificBlue (RM4-5;), anti-MHC I-FitC (KH95), anti-COI Ic-PE (N418;eBioscience, San Diego, Calif., anti-B220-APC (RA3-6B2, eBioscience),anti-COil b-A700 (M 1170), anti-CD II b-FitC (M 1170, eBioscience),anti-CD I Ie-FitC (N418, eBioscienee), anti CD86-A700 (GLI),anti-CD80-PE (16-IOAl), DEC 2OS-APC (20Syekta) and anti-MHC II-Fe(39-10-8,) mAb. All flow cytometey antibodies were purchased from BDPharMingen unless otherwise stated. 50,000-100,000 live events werecollected on a LSRII (BO PharMingen) and analyzed using FlowJo software(Tree Slar, San Carlos, Calif.). The absolute numbers of cells recoveredfrom various organs was determined by multiplying the total number ofcells isolated from various tissues by the percentage of total cellsbearing a lineage specific marker denoted by flow cytometey.

Pancreas RNA Isolation/Histology

Pancreas was harvested from NOD and NOR mice and snap frozen in OCT(Fisher, 14-373-65, Pittsburgh, Pa.) embedding medium in a dewer ofliquid nitrogen and 2-methylbutane (Fisher, 03551-4). Blocks weresectioned on a Leica CM 1950 cryostat at a thickness of 40 microns. RNAwas then isolated using the Arcturus PicoPure RNA Isolation Kit (AppliedBiosystems, KIT0204, Carlsbad, Calif.) and protocol. Purity of RNA wasconfirmed using a Nanodrop ND 1000 Spectrophotometer. Five micronsections were cut and stained with H&E. Photos were taken at 20×magnification using the Leica DM 2500 Microscope equipped with anOptronics color camera and MagnaFire software (Optronics, Goleta,Calif.).

RNA Isolation and RT-qPCR

Total RNA was extracted from the LN and spleens of NOD or NOR mice usingthe SV Total RNA Isolation System (promega, Corp., Madison, Wis., USA),according to the manufacturer's recommended spin column extractionprotocoL The concentrations and purity of the total RNA were determinedusing a SmartSpecPlus Spectrophotometer (BioRad, Hercules, Calif., USA).First-strand cDNA synthesis was performed using ImProm-II ReverseTranscription System (Promega,Corp., Madison, Wis., USA) or iScript RTSupermix for RT-qPCR (BioRad, 170-8841). Absolute QPCR SYBR Green Mix(ABgene Epsom, Surrey, UK) or iQ SYBR Green Supermix Sample (BioRad,170-8880S) and gene specific primers (Table I) al 200 nM were used toamplify relative amounts of cDNA on a PTC-2oo Peltier Thermal Cyclerwith a CHROMO 4 Continuous Fluorescence Detector (BioRad). Amplificationwas performed as previously described (Lau, 112011). The fold-change inexpression was calculated using the double 8CT method (i.e. using theequation T MCT) using BioRad software.

Bacterial DNA Extraction and Analysis

Fresh stool was collected from NOD and NOR mouse strains and frozen at_(—)80° C. Whole DNA was extracted from each stool sample using theQiagen DNeasy Blood and Tissue kit following the manufacturer'sinstructions (Qiagen, Valencia, Calif., USA). Spectrophotometry was usedto determine the DNA concentration and purity of each sample.Amplification and library construction of the V4 region of bacterial J6SrRNA genes was performed using the primers 515F and 806R (3) with theaddition of barcode sequences and required Illumina adapters asdescribed in Fagen et al. (4). For the amplification, an initialdenaturation step of 94° C. for 3 min, followed by 20 cycles of 94° C.for 45 sec, 50° C. for 30 sec, and 65° C. for 90 sec, and a finalelongation step of 65° C. for 10 min was performed. peR products werepurified using the Qiagen™ PCR purification kjt following themanufacturer's protocol (Qiagen, Valencia, Calif., USA).

Illumina Sequencing and Analysis

165 rRNA amplicon sequencing was performed using an lIlumina GAllxsequencing platform (Illumina, Inc., CA, USA) generating IOO×2paired-end reads with an average of 60,409±17,336 reads per sample. Thereads were clustered into operational taxonomic units (OTU) with 97.5%or greater similarity using USEARCH 6.0 (http://www.drive5.comlusearchl)and classified using an RDP database (RDP 10) modified by theTaxCollector program (5). Tables were created and filtered (50 reads fora given OTU in alleast 3 samples; 96.6% of reads were retained) usingLederhosen (httDs:/Igithub.com/audy/lederhosen). Bar graphs andstatistical analyses were conducted in XLSTAT (version 2012.2.01; 2012Addinsoft), an add-in to Microsoft Excel; (version 14.2.5; 2010Microsoft Corporation, Redmond Wash.). Analyses included Spearmancorrelation on taxonomic (proportion of reads) and cytokine messagedata, PCA generation, Shannon diversity index calculation, and I-test ofunequal variance to compare taxonomic abundance between NOD and NORsamples (performed using natural logtransformation of data; p-value˜0.05was considered significant).

Mechanical Separation of Bacteria and Viability Assessment

Bacterial cells were incubated with 0.1 mm glass beads and homogenizedthrough the use of a beadbeater (Qbiogene) according to manufacturers'instructions. Nonviable bacterial components were subsequently separatedthrough ultracentifugation and labeled as membrane, which consisted ofthe pellet, and cytoplasmic, which contained the soluble fraction.Efficiency of bacterial disruption was assessed through the culture ofbacteriallysates on agar plates under anaerobic conditions.

Statistical Calculations

Statistically significant differences were determined using Graph PadPrism software using an unpaired, two-tailed student t test unlessotherwise indicated. Statistical significances are indicated withasterisks symbols: *p<=0.05; **p<=0.0 I; ***p<=0.OO5

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allsolvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Diminished Peripheral Th17 Differentiation and APC Frequencyis Correlated to T1D Onset

In order to better understand the relationship betweenTh17differentiation and T1D onset, IL17 production by stimulated NOD Tlymphocytes was compared to that of NOR lymphocytes. Althoughproliferation and IFNγ production were comparable in activated NOD andNOR peripheral LN suspensions, three-fold more IL17 was produced by NORlymphocytes (FIG. 1A). Since APC derived IL6 is required for TH17differentiation, its production was measured in the NOD and NOR LNsuspensions. Whereas no IL6 production was detected within NOD LNsuspensions, IL6 production was readily observed within the activatedNOR LN suspension (FIG. 1A). Together, these data show that in vitroactivated LN suspensions from diabetes resistant NOR mice possessedhigher levels of Th17 differentiation than pre-diabetic NODcounterparts.

As differences in Th17 differentiation between NOD and NOR LNsuspensions could be due to differences in leukocyte absolute numbers orfrequencies, the cellular composition of the LN were analyzed. CD4+ andCD8+ T lymphocytes were found to be comparable in frequency and absolutenumber between NOD and NOR mice (FIG. 1B). Moreover, CD4+CD25+regulatory T cells, which have been shown to play a critical role in theprevention of T1D onset, were also comparable in number and frequencybetween NOD and NOR peripheral LN (FIG. 2). In stark contrast, NOD LNspossessed significantly fewer APC in both number and frequency (FIG.1B).

Although deficiencies were observed among CD11c+ dendritic cells andCD11b+ macrophages, the most significant deficiencies were observedamong the B220+ B cells (FIG. S1).

Together these data suggest that the reduced capacity of NOD mice toproduce IL17, in comparison to NOR, may be due in part to reductions inAPC function.

EXAMPLE 2 Lymphocytes in Pancreas of NOD Mice Exhibit Reduced Th17 BiasCompared to those Present in NOR Pancreas

NOR and NOD mice experience leukocytic infiltrations of the pancreas,however NOR mice are resistant to insulitis and T1D. Since reducedamounts of IL17 were observed in the peripheral LN of NOD mice comparedto NOR, differences in Th17 associated factors within the leukocyticinfiltrations of the pancreas were studied. Frozen-OCT embedded sectionsfrom the pancreases of both strains were generated, which were processedto either generate H&E stains or to measure the presence of Th17 relatedRNA. Sequential sections were utilized so that infiltrates shown in theH&E histology could be compared to RNA message levels.

Leukocytic infiltrations were observed in pancreatic H&E stains fromboth pre-diabetic 12 week old NOD and age-matched NOR mice (FIG. 3A).The NOD pancreas, however, possessed significantly more T lymphocyteinfiltration and profound insulitis (as denoted by H&E (FIG. 2A) and CD3message (FIG. 3B)).

Although IFNγ levels were indistinct, significantly higher levels ofTh17 associated factors RORγt and IL6 were observed in the NORpancreatic infiltrate compared to that of the NOD (FIG. 3B). IL17message levels were consistently higher in the NOR compared to the NOD.

Together, these data show a reduced Th17 bias within the pancreaticinfiltration of pre-diabetic NOD mice compared to NOR.

EXAMPLE 3 NOD and NOR Mice Possess Distinct Th17 Biases in Mesenteric LNwhich is Correlated to Diverse Microbial Communities

The mesenteric LN of NOR and NOD mice were examined for distinctions incytokine profiles.

Although mesenteric LN cells isolated from NOD mice possessed 50% moreCD3 message, IL17 message levels were significantly lower in the NODmesenteric LN (mLN) compared to the NOR. The Th17 specific transcriptionfactor, RORγt, and surface protein, IL23 receptor (IL23R) were alsoconsistently lower in the NOD mLN (FIG. 4A, 5). Notably, message levelsfor IL23 (required to sustain the Th17 phenotype), but not IL6 wereconsistently lower in the mLN of NOD mice compared to NOR (FIG. 5).

Although lymphocytes present in the gut preferentially track to thepancreatic LN, distinctions in Th17 associated factors were not observedwithin the pancreatic LN (FIG. 6).

Together, these data show that pancreas and mLN of spontaneouslydiabetic NOD mice, possess a significantly lower fraction of Tlymphocytes bearing aTh17 bias when compared to counterpart NOR mice.

In order to analyze the bacterial communities present within NOD and NORmice housed under the same conditions, barcoded 16S rRNA ampliconlibraries were generated from stool samples obtained from the respectivemice and sequenced with Illumina GAIIX. As depicted in FIG. 4B, PCAanalysis revealed that the bacterial composition of NOR mice were moresimilar to other NOR mice than to communities obtained from NOD using97.5% sequence similarity (depicted as weighted distances).

The Shannon diversity index detected no difference in overall communitydiversity between NOD and NOR mice, which was consistent with overallsimilarities observed at the phyla and genus levels (FIG. 7). However,16S rRNA sequencing revealed that the abundance of specific speciespresent in the genera Eubacterium, Clostridium, Syntrophococcus,Bacteroides,and Blautia differed significantly between the bacterialcommunities observed in the stool samples of NOD and NOR mice (FIG. 4Band FIG. 11).

Eubacterium strains E. dolichum and E. ventriosum were observed inhigher frequency in NOD mice, while B. stercoris was 2.5 fold higher(1.0% versus 0.4%) in NOR mice. Although Clostridia were present in bothNOR and NOD mice, notably distinct species were present in each (denotedClostridia (+NOD) versus Clostridia (+NOR), FIG. 4B and Table 1).Additionally, several operational taxonomic units (OTUs) were foundcorrelated with Th17 associated cytokine messages (FIG. 11).

Two Clostridium groups (one unidentified and another closely identifyingto Clostridium alkalicellulosi) were negatively correlated with RORγtmessage and found in greater abundance in NOD mice, while Clostridiumsepticum was found to be positively correlated with IL23R message andmore abundant in NOR mice. Specific Eubacterium, Ruminococcus, andRuminofilibacter species were also correlated with Th17 factors.

Together these data suggest that the enhanced Th17 bias present withinthe pancreas and mesenteric LNs of diabetes resistant NOR mice, comparedto NOD mice, was correlated to distinct microbial communities presentwithin the gut.

EXAMPLE 4 Defects in Th17 Phenotype can be Reversed through APCActivation by Lactobacillus johnsonii N6.2

The oral transfer of a single strain of bacteria, LjN6.2, from diabetesresistant Bio-Breeding Diabetes Resistant (BBDR) rats to Bio-BreedingDiabetes Prone (BBDP) rats was sufficient to confer T1D resistance toBBDP rats (Valladares R, et al 2010). Moreover, the conferred T1Dresistance was correlated to an APC dependent, Th17 bias observed in theBBDP rat and the NOD mouse (Lau K, et al 2011). Conversely, a secondcommensal bacterial strain, LrTD1, neither conferred T1D resistance normediated a Th17 bias (Valladares R, et al 2010; Lau K, et al 2011). Thecapacity of LjN6.2 to modulate APC was compared to LrTD1 in vitro.Treatment of NOD derived primary cell cultures with LjN6.2, but notLrTD1, consistently up-regulated the frequency of both CD11b+ and CD11c+leukocytes in a dose dependent manner (FIG. 8A, B). Although thefrequencies of both CD11b+ and CD11c+ leukocytes present in NOD culturesincreased in a dose dependent manner, CD11b+ and CD11c+ leukocytes inNOR cultures were only moderately increased (FIG. 4B).

The capacity of LjN6.2 to modulate APC cell activation was examined.LjN6.2 treatment mediated a decrease in CD11b+MHCIhi leukocytes and anincrease in the frequency CD11b+MHCIIhi leukocytes in both NOD and NORcultures (FIG. 8C). Notably, although NOD derived CD11c+ dendriticcells, treated with LjN6.2, upregulated levels of MHC classes I and IIin a dose dependent manner, LjN6.2 failed to specifically increase MHCIand MHCII expression in NOR derived CD11c+ DC (FIG. 8C). Significantly,in contrast to LjN6.2, LrTD1 treatment mediated reduced, or negligible,modulation of MHC (FIG. 8C).

Together these data show that LjN6.2, but not LrTD1, modulates the Tcell priming capacity of NOD derived APC through increased frequency andup-regulation of MHC surface expression.

EXAMPLE 5 LjN6.2 Mediates Endocytic Marker, DEC205 Down-Modulation onDendritic Cells

The capacities of LjN6.2 and LrTD1 to modulate DEC205 levels ondendritic cells was measured. LjN6.2, but not LrTD1, mediateddown-regulation of DEC205 expression in both NOD and NOR CD11c+dendritic cells in a dose dependent manner (FIG. 9). Notably, althoughLjN6.2 did not up regulate MHC molecules on NOR derived CD11c+leukocytes, it did mediate DEC205 down modulation.

These data suggest that LjN6.2 interactions with DEC205 helped tomediate the capacity of dendritic cells to prime Th17 effectorfunctions. LjN6.2 surface antigen modulates dendritic cell function.

IL6 production by BMDCs incubated with either viable LjN6.2, orbacterial cellular equivalents of bead beater ruptured LjN6.2 wasmeasured. The bead beater ruptured LjN6.2 components were separated bycentrifugation into insoluble (designated membrane) or soluble(designated cytoplasmic) components. BMDC incubated with LjN6.2 producedcopious amounts of IL6 (FIG. 9C). Significantly, lysed LjN6.2 alsomediated significant amounts of IL6 in a dose dependent manner (FIG. 9Cand FIG. 10). In contrast, the cytoplasmic portion of the bacteriafailed to up-regulate IL6 production. It is highly unlikely that the IL6production is due to residual viable bacteria that survived the lysisprocess as we were unable to detect bacterial colonies from platesstreaked with membrane components in contrast to plates streaked withLjN6.2 at the same initial bacterial concentration (FIG. 9C). It wasalso confirmed that IL6 and IL17 production could be mediated by LjN6.2cell membrane components, but not the cytoplasmic components, in a dosedependent manner (FIG. 10).

Together, these data strongly suggest that components present on thesurface of LjN6.2 specifically modulate the T cell priming capacity ofdendritic cells, possibly through interactions with DEC205.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

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We claim:
 1. A composition for treating or preventing type 1 Diabetes (T1D), the composition comprising an antigen presenting cell (APC) pulsed with a bacterium or a component of the bacterium, wherein the bacterium or the component of the bacterium confers upon the APC the ability to inhibit the generation of diabetes promoting T cells.
 2. The composition of claim 1, wherein the APC is a dendritic cell.
 3. The composition of claim 1, wherein the bacterium is selected from the genera Eubacterium, Clostridium, and Lactobacillus.
 4. The composition of claim 3, wherein the bacterium is Lactobacillus johnsonii or Lactobacillus johnsonii N6.2.
 5. The composition of claim 1, wherein the component of the bacterium is a membrane component.
 6. The composition of claim 1, wherein the bacterium is nonviable.
 7. A method of treating or preventing T1D, the method comprising administering to a subject having T1D or having an increased risk of developing T1D, a composition comprising an effective amount of an APC pulsed with a bacterium or a component of the bacterium, wherein the bacterium or the component of the bacterium confers upon the APC the ability to inhibit the generation of diabetes promoting T cells in the subject.
 8. The method of claim 7, wherein the APC is DC.
 9. The method of claim 7, wherein the bacterium is selected from the genera Eubacterium, Clostridium, and Lactobacillus.
 10. The method of claim 7, wherein the bacterium is Lactobacillus johnsonii or Lactobacillus johnsonii N6.2.
 11. The method of claim 7, wherein the component of the bacterium is a membrane component.
 12. The method of claim 7, wherein the bacterium is nonviable.
 13. A method of preparing a vaccine to treat or prevent T1D, the method comprising pulsing an APC with a bacterium or a component of the bacterium, wherein the bacterium or the component of the bacterium confers upon the APC the ability to inhibit the generation of diabetes promoting T cells.
 14. The method of claim 13, wherein the APC is a DC.
 15. The method of claim 13, wherein the bacterium is selected from the genera Eubacterium, Clostridium, and Lactobacillus.
 16. The method of claim 13, wherein the bacterium is Lactobacillus johnsonii or Lactobacillus johnsonii N6.2.
 17. The method of claim 13, wherein the component of the bacterium is a membrane component.
 18. The method of claim 13, wherein the bacterium is nonviable. 